Kirkwood A. Pritchard

Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression.

Recently, we have shown that chronic exercise increases endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-mediated epicardial coronary artery dilation in response to brief occlusion and acetylcholine. This finding suggests that exercise can provide a stimulus for the enhanced production of EDRF/NO, thus possibly contributing to the beneficial effects of exercise on the cardiovascular system. Therefore, the purpose of the present study was to examine whether chronic exercise could influence the production of NO (measured as the stable degradation product, nitrite) and endothelial cell NO synthase (ECNOS) gene expression in vessels from dogs after chronic exercise. To this end, dogs were exercised by running on a treadmill (9.5 km/h for 1 hour, twice daily) for 10 days, and nitrite production in large coronary vessels and microvessels and ECNOS gene expression in aortic endothelial extracts were assessed. Acetylcholine (10(-7) to 10(-5) mol/L) dose-dependently increased the release of nitrite (inhibited by nitro-L-arginine) from coronary arteries and microvessels in control and exercised dogs. Moreover, acetylcholine-stimulated nitrite production was markedly enhanced in large coronary arteries and microvessels prepared from hearts of dogs after chronic exercise compared with hearts from control dogs. One potential mechanism that may contribute to the enhanced production of nitrite in vessels from exercised dogs may be the induction of the calcium-dependent ECNOS gene. Steady-state mRNA levels for ECNOS were significantly higher than mRNA levels for von Willebrands factor (vWF, a specific endothelial cell marker) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a constitutively expressed gene) in exercised dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion

To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (·NO) and superoxide anion (O⨪2) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ·NO. As eNOS is capable of generating both ·NO and O⨪2, we hypothesized that Hsp90 might also mediate eNOS-dependent O⨪2 production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 μg/ml; 17.8 μm) and then stimulated with the calcium ionophore,A23187 (5 μm). GA significantly decreasedA23187-stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased A23187-stimulated eNOS-dependent O⨪2production (p < 0.001, n = 8).A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O⨪2production.

Reduced Gene Expression of Vascular Endothelial NO Synthase and Cyclooxygenase-1 in Heart Failure

Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures > 25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with 32P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66 +/- 0.77 (mean +/- SEM, n = 17) and 1.12 +/- 0.37 (n = 6 and the ratios of COX-1 to GAPDH were 1.52 +/- 0.52 and 0.56 +/- 0.15 before and after heart failure, respectively. These represent 56% to 64% (P < .05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (P < .05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production.

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were “uncoupling” NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.

Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study.

©1997 Federation of European Biochemical Societies.

TETRAHYDROBIOPTERIN-DEPENDENT INHIBITION OF SUPEROXIDE GENERATION FROM NEURONAL NITRIC OXIDE SYNTHASE

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH4) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH4 analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields.l-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity forl-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with l-arginine. This effect is in accordance with the slow binding rates ofl-arginine to NOS in the absence of BH4.l-Arginine alone or in combination with BH4decreases the rates of NADPH consumption. The effect ofl-arginine on superoxide yields, however, was less dramatic than that caused by BH4 as much higher concentrations ofl-arginine are necessary to attain the same inhibition. In combination, l-arginine and BH4 inhibit the formation of superoxide generation and stimulate the formation ofl-citrulline. We conclude that, in contrast tol-arginine, BH4 does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.

L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease

Background—Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)–dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2·−) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor–null (Ldlr−/−) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2·− generation. Methods and Results—Arterioles were isolated from hypercholesterolemic Ldlr−/− mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2·− generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase–inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr−/− mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2·− in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. Conclusions—These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Suppression of Angiotensin-Converting Enzyme Expression and Activity by Shear Stress

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.

Erythropoietin protects the infant heart against ischemia–reperfusion injury by triggering multiple signaling pathways

Abstract The immediate protective effect of erythropoietin (EPO) against ischemia in heart suggests a role beyond hematopoiesis and the treatment of anemia. We determined the role of JAK/STAT and Ras/Rac/MAPK in the protective effect of EPO against ischemia–reperfusion injury in infant rabbit heart. EPO (1.0 U/ml) administered 15 minutes prior to 30–minutes global ischemia and 35 minutes reperfusion resulted in increased recovery of postischemic ventricular developed pressure in rabbit hearts. EPO exerted its immediate cardioprotective effect via activation of multiple signaling pathways by: 1) phosphorylation and activation of JAK1/2, STAT3 and STAT5A but not of STAT1α and STAT5B, 2) phosphorylation and activation of PI3 kinase and its downstream kinases Akt and Rac, 3) activation of PKCε, Raf, MEK1/2, p42/44 MAPK and p38 MAPK. Pretreatment with Wortmannin abolished EPO–induced Akt activation and phosphorylation. Pretreatment with Chelerythrine followed by EPO treatment resulted in partial inhibition of Raf activation, and abolished PKCε and p38 MAPK activation without any effect on Akt, MEK1/2 and p42/44 MAPK. PD98059 abolished MEK1/2 and p42/44 MAPK activation with no effect on Akt, Raf and p38 MAPK activation. SB203580 inhibited only p38 MAPK activation by EPO. We can conclude EPO increases immediate cardioprotection through the activation of multiple signal transduction pathways.